Bottom Line:
In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1.Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1).Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation.

Affiliation: Graduate Program in Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachussets, United States of America.

ABSTRACTNecroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNFα production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNFα. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNFα production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation.

pone-0056576-g008: Akt signaling contributes to autocrine TNFα production in multiple cell types.FADD deficient Jurkat cells were treated with TNFα followed by measurement of (A) human TNFα mRNA levels by qRT-PCR and normalized using human 18S RNA or (B) western blot at 9 hr. RAW 264.7 or J774A.1 cells were treated with zVAD.fmk (100 uM or 50 uM respectively) followed by (C,E) measurement of TNFα mRNA levels by qRT-PCR or (D,F) western blot at 9 hr. (G) Akt mouse lung fibroblasts expressing Myr-Akt or K179M were treated with zVAD.fmk and TNFα followed by measurement of TNFα mRNA levels by qRT-PCR at 9 hr. (H) Mouse lung fibroblasts expressing only endogenous Akt1 or Akt2 were treated with zVAD.fmk and TNFα followed by measurement of TNFα mRNA levels by qRT-PCR at 9 hr.

Mentions:
After establishing the role of RIP1 kinase-dependent signaling to Akt in L929 cells, we sought to expand our study to other cell types that are known to undergo necroptotic cell death. Fas-associated protein with death domain (FADD)-deficient Jurkat T lymphocytes and the macrophage cell lines (J774A.1 and RAW264.7) are other models of necroptosis, which can be induced by stimulation with TNFα or zVAD.fmk, respectively [17]. Similar to L929 cells, a RIP1 kinase dependent increase in the phosphorylation of Thr308 on Akt occurred during necroptosis (Fig. 8B,D,F) in these cell types. Furthermore, TNFα mRNA levels were increased in each of these cell types during necroptosis and efficiently inhibited by both RIP1 and Akt inhibitors (Fig. 8A,C,E). However, inhibition of Akt did not protect these cells from death (Fig. S9A,B,C). These results indicate that regulation of autocrine TNFα synthesis and necroptosis-associated inflammatory signaling may be a more important function of Akt pathway activation by RIP1 kinase in multiple cell types compared to its contribution to cell death.

pone-0056576-g008: Akt signaling contributes to autocrine TNFα production in multiple cell types.FADD deficient Jurkat cells were treated with TNFα followed by measurement of (A) human TNFα mRNA levels by qRT-PCR and normalized using human 18S RNA or (B) western blot at 9 hr. RAW 264.7 or J774A.1 cells were treated with zVAD.fmk (100 uM or 50 uM respectively) followed by (C,E) measurement of TNFα mRNA levels by qRT-PCR or (D,F) western blot at 9 hr. (G) Akt mouse lung fibroblasts expressing Myr-Akt or K179M were treated with zVAD.fmk and TNFα followed by measurement of TNFα mRNA levels by qRT-PCR at 9 hr. (H) Mouse lung fibroblasts expressing only endogenous Akt1 or Akt2 were treated with zVAD.fmk and TNFα followed by measurement of TNFα mRNA levels by qRT-PCR at 9 hr.

Mentions:
After establishing the role of RIP1 kinase-dependent signaling to Akt in L929 cells, we sought to expand our study to other cell types that are known to undergo necroptotic cell death. Fas-associated protein with death domain (FADD)-deficient Jurkat T lymphocytes and the macrophage cell lines (J774A.1 and RAW264.7) are other models of necroptosis, which can be induced by stimulation with TNFα or zVAD.fmk, respectively [17]. Similar to L929 cells, a RIP1 kinase dependent increase in the phosphorylation of Thr308 on Akt occurred during necroptosis (Fig. 8B,D,F) in these cell types. Furthermore, TNFα mRNA levels were increased in each of these cell types during necroptosis and efficiently inhibited by both RIP1 and Akt inhibitors (Fig. 8A,C,E). However, inhibition of Akt did not protect these cells from death (Fig. S9A,B,C). These results indicate that regulation of autocrine TNFα synthesis and necroptosis-associated inflammatory signaling may be a more important function of Akt pathway activation by RIP1 kinase in multiple cell types compared to its contribution to cell death.

Bottom Line:
In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1.Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1).Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation.

Affiliation:
Graduate Program in Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachussets, United States of America.

ABSTRACTNecroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNFα production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNFα. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNFα production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation.